New insights into Bacillus cytotoxicus sources, screening, toxicity, and persistence in food production facilities.

Bacillus cytotoxicus is a thermotolerant member of the Bacillus cereus group. It has been linked to rare, but at times fatal cases of diarrheal disease and might be missed at routine diagnostic screening temperatures commonly used for the B. cereus group. The pathogen is mostly found on dehydrated foods containing potato starch or insects. How it enters the food chain or whether it persists in food producing environments is largely unknown. Increased consumption of insects and convenience foods in Europe and the lack of information on the persistence of B. cytotoxicus in food environments and its virulence demand for further characterization. In this study, we aimed to obtain a better understanding of i) the food sources of B. cytotoxicus, ii) screening temperatures needed for its isolation from food matrices, iii) cytotoxicity of the organism, and iv) its ecological niche and potential epidemiological links. To this end, 112 food samples were collected, with a focus on foods exhibiting low water activity. The samples were screened for B. cytotoxicus at 42 °C and at 50 °C. Presumptive isolates were characterized by cytK-1 toxin gene PCR for differentiation of B. cytotoxicus from other B. cereus group members. Vero cell cytotoxicity assays were performed, and selected isolates were sequenced. Our results show that screening at 42 °C might be insufficient for detecting B. cytotoxicus in foods that harbor other less thermophilic Bacillus species. When screening at 50 °C, B. cytotoxicus was detected in 23% of the food samples (n = 26 isolates). The highest prevalence was detected in mashed potato products (82%) and potato flakes (67%). In contrast, a wide range of products not containing any potato ingredients did not yield B. cytotoxicus isolates. All B. cytotoxicus isolates exhibited either low or no detectable cytotoxicity. WGS analysis revealed that a highly toxic isolate is closely related to the French outbreak strain NVH 391-98. In addition, we could show that two isolates sampled 5 years apart from the same production facility only differed by seven SNPs, making it likely that B. cytotoxicus is able to persist in production facilities over a long time. Interestingly, the reoccurring strain possessed an additional plasmid and did not show cytotoxic potential when re-isolated after 5 years.

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